Challenges in EGFRvIII detection in head and neck squamous cell carcinoma

Abstract

Objective: Head and neck squamous cell carcinoma (HNSCC) accounts for more than 5% of all cancers worldwide. The mortality rate of HNSCC has remained unchanged (approximately 50%) over the last few decades. Ubiquitous overexpression of wild type EGFR in many solid tumors has led to the development of EGFR targeted therapies. EGFR can be constitutively activated via several mechanisms including the truncated, EGFR variant III isoform (EGFRvIII). EGFRvIII lacks exons 2-7 and has been reported to be present in up to 20-40% of HNSCC. EGFRvIII has been shown to contribute to cetuximab resistance. The mechanisms leading to EGFRvIII expression in HNSCC are unknown. The present investigation was undertaken to determine the etiology of EGFRvIII in HNSCC.

Materials and methods: Fixed HNSCC and glioma tissues were analyzed by fluorescence in situ hybridization for EGFR amplification. DNA and RNA from fresh frozen specimens were used to determine the presence of EGFRvIII transcripts and the mechanisms of expression via PCR, RT-PCR and RNA sequencing.

Results: Unlike glioma, EGFRvIII expression in HNSCC did not correlate with EGFR amplification. We found evidence of genomic deletion of the exon 2-7 in 6 of 7 HNSCC cases examined, however, the presence of genomic deletion did not always result in mRNA expression of EGFRvIII. RNA sequencing with automated alignment did not identify EGFRvIII due to microhomology between intron 1 and exon 8. RNA sequencing analyzed by manual alignment methods did not correlate well with RT-PCR and PCR findings.

Conclusion: These findings suggest that genomic deletion as well as additional regulatory mechanisms may contribute to EGFRvIII expression in HNSCC. Further, large scale automated alignment of sequencing are unlikely to identify EGFRvIII and an assay specifically designed to detect EGFRvIII may be necessary to detect this altered form of EGFR in HNSCC tumors.

Fig 1. EGFRvIII correlation with EGFR amplification and HPV.

A. Twenty five HNSCC tumors with known EGFR gene amplification status (via FISH) were tested for EGFRvIII positivity (28 tumors are shown, three tumors marked with N did not have gene amplification data). RNA was isolated and EGFRvIII and GAPDH were RT-PCR amplified as described in Materials and Methods. 4 of 12 EGFR amplified samples contained EGFRvIII, 5 of 12 of samples without EGFR amplification expressed EGFRvIII. All EGFRvIII bands were excised and sequenced to verify exon 1 to exon 8 joining (samples with confirmed EGFRvIII are denoted by “+”). B. A fisher’s exact test showed a lack of association between EGFRvIII expression and EGFR amplification (p = 0.56). C. A fisher’s exact test showed a lack of association between EGFRvIII expression and p16 expression (p = 0.27).

Fig 2. PCR amplification of EGFR for genomic alterations leading to EGFRvIII transcript.

A. Schematic of sequencing primers and areas of interest. Arrows indicate the location of the primers used to detect EGFRvIII in genomic DNA and unspliced RNA. Bars with diamond caps indicate areas amplified for splice donor and acceptor mutations. The shaded area is lost in EGFRvIII. B. Representative PCR amplification of the splice donor/acceptor sites of EGFR exons 1, 2, 7 and 8 in an EGFRvIII positive HNSCC DNA sample. These bands were excised and sequenced for mutations. C. Representative long-range PCR amplification of EGFR intron 1 for a single EGFRvIII positive HNSCC DNA sample. L: base pair marker, W: water control, a-j: primer sets.